Abstract

The bacterial twin-arginine translocation (Tat) pathway is able to export pre-foldedcofactor containing proteins across the cytoplasmic membrane. Tat substrates bearcleavable N-terminal signal peptides that are characterized by the presence of acritical and highly conserved twin-arginine motif which lends the Tat pathway itsname.In Escherichia coli and many other Gram-negative bacteria, three integral membraneproteins: TatA, TatB and TatC are essential for Tat-dependent translocation. Incontrast Bacillus subtilis possesses a simpler TatAC system which lacks the TatBcomponent. In E. coli the TatA protein assembles into homo-oligomeric complexesthat vary considerably in size. The TatA proteins found in B. subtilis do not exhibitthe same degree of heterogeneity and this suggested mechanistic differences betweenthe Tat pathways of Gram-negative and Gram-positive bacteria. How the Tat systemworks is still poorly understood, and the work presented in this thesis sought to gaininsights into the assembly and mechanism of E. coli and B. subtilis Tat pathways.This work focused on the study of two previously uncharacterized components: theE. coli TatA paralog TatE subunit and B. subtilis TatAc subunit.In this thesis the purification and characterization of E. coli TatE complexes isreported. Using analytical gel filtration chromatography, blue-native gelelectrophoresis(BN-PAGE) and single-particle analysis of purified TatE complexes,it was found that the TatE complexes are more discrete than the highlyheterogeneous TatA complexes. This finding, together with the ability of TatE tosupport the translocation of the 90-kDa TorA protein, suggested alternativetranslocation models in which single TatE complexes do not contribute the bulk ofthe translocation channel, similar to the B. subtilis model.In addition, co-purification and BN-PAGE experiments demonstrated for the firsttime that TatE interacts with TatA to form TatAE mixed complexes in themembrane, and reveals a completely novel form of Tat complex that might befunctionally significant.A soluble population of TatE was also identified in E. coli cell extracts, and phaseseparation experiments using Triton X-114 suggested it may be mis-localized.In a separate set of studies, the ability of the B. subtilis TatAc protein to form activetranslocases in combination with the B. subtilis TatCd or TatCy proteins wasinvestigated for the first time. The TatAcCd and TatAcCy mixed translocases wereable to translocate several E. coli Tat substrates including, TorA, AmiA and AmiC.Finally BN-PAGE and gel filtration chromatography showed that the TatAcCd andTatAcCy complexes were significantly smaller than the previously described E. coliTatABC substrate-binding complex.